Nucleic acid hybridization, the formation of a double strand of nucleic acids by formation of hydrogen bonds between complementary strands of nucleic acids, is a well known phenomenon finding increasing application. For example, hybridization is the core phenomenon of so-called "genetic probe" assays. Thus, an assay to confirm the presence in a suitably prepared sample of a nucleic acid of diagnostic significance can be built around a nucleic acid, usually an oligonucleotide (oligomer) of known sequence (the "probe") which is complementary to a nucleotide sequence within the targeted nucleic acid. See, for example, U.S. Pat. No. 4,358,535.
The formation of hybrid between the probe and targeted nucleic acid, usually detected by a suitable label linked to the probe after separation of unbound probe, is taken as confirmation that the complementary sequence is present in the nucleic acid of the sample. The presence of this sequence, if properly selected, permits inferences of diagnostic significance to be drawn. For example, if the sequence complementary to the probe is unique to a genus or species of bacteria which causes disease, the presence of the genus or species in the sample is confirmed if hybrid formation is detected. Absence of hybrid formation, on the other hand, permits the negative inference, i.e., that the sample does not contain the suspect organism (or organisms), at least within the detection limits of the assay. Such an assay might be used with other symptomatology to diagnose that disease is present or that disease is caused by the detected organism. A similar assay might show its presence in food intended for human consumption. Using similar techniques, nucleic acid probes can be used to detect not only bacteria, but also disease causing fungi, viruses, oncogenes or protooncogenes, genes associated with a variety of genetic diseases, and the like.
It has also been proposed to use nucleic acid probes therapeutically. For example, if a cell is infected by a virus, and a probe is introduced into the cell which is complementary to at least a portion of the messenger RNA (mRNA) encoded by the virus or to its genomic nucleic acid, binding of the probe to the targeted viral nucleic acid would prevent its transcription or translation by the cell's ribosomes, effectively preventing the virus from replicating. This phenomenon of probe hybridization with mRNA is referred to as "hybridization arrest." Hybridization arrest using a methylphosphonate derivative of DNA as a probe is described in U.S. Pat. No. 4,511,713. The use of hybridization arrest techniques to inhibit mRNA translation of dihydrofolate reductase in an in vitro model using anti-sense oligonucleotides, including mixtures of short oligonucleotide sequences, is described by Maher, et al., Archives of Biochem. and Biophys., 253, 214-20 (1987).
In the case of using genetic probes to detect organisms, particularly infectious organisms, it has heretofore been the general practice to target DNA in order to identify the organisms of interest. Because DNA is already double stranded, this has made it necessary to not only lyse the cell to liberate the DNA, but also to denature (melt) the double stranded DNA to obtain a single stranded structure. This is typically done by heating the double stranded DNA to a temperature at which the duplex structure comes completely apart. The temperature at which this occurs in solution can vary. The T.sub.m of a duplex (the temperature at which 50% of the strands of DNA have separated) is increased by increasing the ionic strength of the solution and decreases in the presence of reagents such as formamide which destabilize hydrogen bonds.
After denaturation, the DNA is typically fixed to a solid surface such as nitrocellulose to preclude reformation of the DNA's binary structure (renaturation) by hybridization of the separated strands. See U.S. Pat. No. 4,358,535. While fixing the DNA to a solid surface prevents renaturation, it imposes heterogeneous kinetics, with their attendant disadvantages including a much slower rate of hybridization, on the assay system. Fixing the DNA to a solid surface also may fix the DNA in an orientation which prevents hybridization with the probe.
To overcome these limitations it has been proposed to conduct hybridization in solution since solution kinetics are much more favorable than heterogeneous kinetics. As a result, hybridization goes to completion in solution much faster than would be the case if the targeted DNA is fixed to a solid surface. In-solution hybridization can be carried out by adding probe to the denatured DNA and reestablishing conditions under which duplex formation can occur. If a sufficient excess of probe is used, it can compete effectively for the particular nucleotide sequence in the targeted nucleic acid to which the probe is directed with the DNA present in the sample that is complementary to that sequence.
At least some of the problems associated with targeting DNA can be avoided by using RNA as a target. RNA is already single stranded and, therefore, the necessity for denaturing and fixing the DNA to a solid phase or carrying out hybridization under conditions in which the probe must compete with the organism's own DNA is eliminated. In the case of viruses, mRNA can be a useful target. However, in the case of pro-and eucaryotes, it is preferred to target ribosomal RNA (rRNA) since each cell contains about 10.sup.3-10.sup.4 as much rRNA target sites as genomic DNA. Thus targeting rRNA, if available as a target, permits assays of much greater sensitivity.
Assay methods which target RNA and exploit in-solution hybridization are described in Canadian Patent No. 1,215,904 and European Patent Application No. 84900667.1, the disclosures of which are incorporated herein by reference.
Although much more convenient for the user, the development of assays which target ribosomal RNA presents problems. Often a candidate probe, which otherwise appears to be ideal, fails because it exhibits a very slow reaction rate or poor extent of reaction, even when hybridization is carried out in solution. As a result, it may in some cases be necessary to select for an assay a probe which compromises specificity in order to achieve the desired kinetics. In other cases, it may be necessary to sacrifice sensitivity in order to achieve a commercially viable assay because of the slow kinetics or poor extent of hybrid formation.
Approaches to accelerating the rate of hybridization of complementary nucleotide multimers have been explored. Among those are the addition of nucleic acid precipitating reagents to the hybridization solution as described in application for U.S. application Ser. No. 57,981, filed June 4, 1987, assigned to the assignee of this application and the disclosure of which is incorporated by reference as if fully set forth herein.
The use of rate acceleration techniques as described in the above-referenced application does not in every case provide a rate of hybridization increase which permits optimal assay development. As a result, there remains a need for other means of enhancing the kinetics of hybridization between a probe and its target sequence which can be used with, or even in lieu of, other techniques for accelerating the rate of hybridization between complementary nucleotide multimers.
Another problem is sometimes encountered in the development of assays of narrow specificity, particularly when the assay is directed to a single species of an organism in a genus containing closely related species. The sequence homology of the genomic DNA and ribosomal RNA of the target species and its close relatives is very close in such cases and these nucleic acids often contain mismatches of only one or two nucleotide bases in relatively long sequences.
With the advent of nucleic acid synthesizers, it has been possible to design and synthesize probes which are a perfect, or near perfect, complement for a sequence in the targeted nucleic acid. The T.sub.m of a hybrid between the probe and its complement in the targeted nucleic acid is a function of the number of complementary nucleotides involved in hybrid formation, i.e., as the length of the probe increases so generally does the T.sub.m. Therefore, the probe must be long enough so that a stable hybrid is formed at the temperature at which the assay is carried out.
This temperature is selected so that the extent of hybridization within a reasonable time is enough to give the assay adequate sensitivity. Often a probe long enough to permit this has sufficient complementarity with a sequence in one or more closely related species that significant hybridization with the nucleic acid of the closely related species can also occur during the assay. This cross reactivity is usually due to the fact that their melting profiles are overlapping. This can reduce the specificity of the assay by causing false positive results. Cross reactivity could be significantly reduced or even avoided, however, if the T.sub.m of relatively short probes could be raised since the difference in T.sub.m between a hybrid of a probe and its perfect match and a hybrid of the probe with a nucleic acid having one or more nucleotide mismatches is usually greater for a short probe compared to a larger one having the same number of mismatches. The shorter probe has a higher percentage of mismatches to its nearest neighbor than the longer probe which can result in their melting profiles no longer overlapping. This larger difference in the T.sub.m means that the mismatched hybrid can be completely dissociated while the percentage of hybridization of probe to target remains high. Reduction in such cross reactivity would, of course, have the result of reducing or eliminating false positive results with a resulting increase in assay specificity.